CD RNA Multiplex Oligos S1 for Illumina was created specifically for the preparation of CD RNA libraries on Illumina platforms. CD RNA Adapter-S for Illumina, 8 types of 8bp indexed i5 and 12 types of 8bp i7 are included in the kit. Different samples can be distinguished using the dual-indexed adapter. It contains adaptors and primers that allow for high-yield multiplex Illumina library production as well as index incorporation during library amplification. High adaptor ligation efficiency, minimal adaptor-dimer formation, and index primers for library multiplexing are some of the advantages of this kit.
Components:
Application:
Applicable for dual-indexed RNA library preparation for Illumina® platforms with CD mRNA-seq Library Prep Kit for Illumina (RP001), CD Stranded mRNA-seq Library Prep Kit for Illumina (RP002) or CD Total RNA-seq Library Prep Kit for Illumina(human/mouse/rat) (RP003).
Storage:
All the components can be stored at -20℃ for one year.
Specifications:
| Application | Used for: CD mRNA-seq Library Prep Kit for Illumina CD Stranded mRNA-seq Library Prep Kit for Illumina CD Total RNA-seq Library Prep Kit for Illumina(human/mouse/rat) |
| Sequencing Platform | Illumina |
Adapter Ligation
- Bring RNA Adapter from -20° and mix it thoroughly after thawing.
- Invert thawed Rapid Ligation buffer and place it on ice.
- Combine the reaction in a sterile PCR tube (please refer to the product handbook for the components of the reaction mixture).
- Mix the reaction by gently pipette for 10 times.
- Place the reaction tube in the PCR instrument to perform ligation reaction.
Purification and Size-selection of Ligation Product
Please refer to the following table if size selection is needed:
| Insertion Length (bp) | 200-300 | 250-350 | 450-550 | 350-450 |
| Library Length (bp) | 260-360 | 310-410 | 510-610 | 410-510 |
| Fragmentation Condition | 94°C 5 min | 85°C 6 min | 85°C 5 min | 85°C 6 min |
| 1st Round bead volume (μl) | 80 (0.8 ×) | 65 (0.65 ×) | 55 (0.55 ×) | 60 (0.6 ×) |
| 2nd Round bead volume (μl) | 20 (0.2 ×) | 20 (0.2 ×) | 10 (0.1 ×) | 10 (0.1 ×) |
Library Amplification
- Invert the thawed PCR Primer Mix and combine the reaction below in a sterile PCR tube.
- Place the reaction tube in the PCR instrument and operate under the condition that is provided in the product handbook.
Sequence Information
RNA Adapter-S for Illumina
5´-ACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´
3´-CTGACCTCAAGTCTGCACACGAGAAGGCTAG-p-5´
(-s- indicates Sulfo; -p indicates Phosphorylation.)
i5 PCR Primers
5´-AATGATACGGCGACCACCGAGATCTACAC[i5]ACACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´ i7 PCR Primers
5´-CAAGCAGAAGACGGCATACGAGAT[i7]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´
(-s- indicates Sulfo.)
[i5] indicates i5 Index sequence (8 bp);[i7] indicates i7 Index sequence (8 bp).
Detailed information is as follows:
| i5 PCR Primers | Name of i5 Index | Index Sequence in Primer | Index Sequence for Sample | Sheet Input/ for Sequencing |
| NovaSeq, MiSeq, HiSeq 2000/2500 | MiniSeq, NextSeq, HiSeq 3000/4000 | |||
| RM501 | R501 | TATAGCCT | TATAGCCT | AGGCTATA |
| RM502 | R502 | ATAGAGGC | ATAGAGGC | GCCTCTAT |
| RM503 | R503 | CCTATCCT | CCTATCCT | AGGATAGG |
| RM504 | R504 | GGCTCTGA | GGCTCTGA | TCAGAGCC |
| RM505 | R505 | AGGCGAAG | AGGCGAAG | CTTCGCCT |
| RM506 | R506 | TAATCTTA | TAATCTTA | TAAGATTA |
| RM507 | R507 | CAGGACGT | CAGGACGT | ACGTCCTG |
| RM508 | R508 | GTACTGAC | GTACTGAC | GTCAGTAC |
| i7 PCR Primers | Name of i7 Index | Index Sequence in Primer | Index Sequence for Sample Sheet Input/ for Sequencing |
| RM701 | R701 | CGAGTAAT | ATTACTCG |
| RM702 | R702 | TCTCCGGA | TCCGGAGA |
| RM703 | R703 | AATGAGCG | CGCTCATT |
| RM704 | R704 | GGAATCTC | GAGATTCC |
| RM705 | R705 | TTCTGAAT | ATTCAGAA |
| RM706 | R706 | ACGAATTC | GAATTCGT |
| RM707 | R707 | AGCTTCAG | CTGAAGCT |
| RM708 | R708 | GCGCATTA | TAATGCGC |
| RM709 | R709 | CATAGCCG | CGGCTATG |
| RM710 | R710 | TTCGCGGA | TCCGCGAA |
| RM711 | R711 | GCGCGAGA | TCTCGCGC |
| RM712 | R712 | CTATCGCT | AGCGATAG |
